This proposal describes strategies to synthesize potent non-competitive inhibitors of botulinum neurotoxin metalloproteases {BoNT protease}. These metalloproteases which are the molecular effectors of the paralytic effects of these toxins are the most selective proteases reported due to allosteric activation of the enzyme by a portion of the substrate. Conformationally-restricted analogs of known substrate "activation" sequences will be synthesized to develop peptide- derived inhibitors that inhibit at a non-competitive site on the enzyme. Yeast two-hybrid experiments will be used to identify the enzyme residues at the activation site. When the bioactive conformation has been elucidated, it will become possible to develop non-peptide templates for creating novel inhibitors of these proteases. During Phase I, the applicants will synthesize a set of conformationally restricted compounds and determine their inhibitory activity using the established assay. Known substrates are cleaved by the toxin metalloprotease to generate fragments with an electrophoretic mobility different from those of the uncleaved substrates. These compounds are expected to function as pharmacological antagonists and to serve as important probes useful for neurobiological research as adjuncts for reversing effects of BoNT in treating achalasia and related disorders in humans and as important lead compounds in antitoxin drug development.